《植物生理学报》 2016, 52(8): 1223-1230

水稻水通道蛋白OsPIP1;3与白叶枯病菌harpin蛋白Hpa1互作关系研究

王欢, 田沂民, 董汉松^*

南京农业大学植物保护学院, 南京210095

通信作者：董汉松；E-mail: hsdong@njau.edu.cn

摘　要：

本文通过片段缺失和定点突变的方法构建产生水稻(Oryza sativa)水通道蛋白OsPIP1;3的变异本。通过膜酵母双杂交进行蛋白互作分析, 实验结果表明水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae) harpin蛋白Hpa1能与OsPIP1;3互作, 但如果将OsPIP1;3的一个胞外序列(loop E)及OsPIP1;3的第六个跨膜区域(TM6)删除, 则互作不能发生。进一步对276~279 aa进行替换, OsPIP1;3与Hpa1不再发生互作。这些结果说明, OsPIP1;3序列上的loop E和TM6上的氨基酸对OsPIP1;3–Hpa1互作有重要影响, 而276~279 aa是可能的互作位点。

关键词：Hpa1; OsPIP1;3; 定点突变; 膜酵母双杂交; 蛋白互作

收稿：2016-03-28   修定：2016-06-21

资助：公益性行业(农业)科研专项(201303015)。

Studies on the interaction between aquaporin OsPIP1;3 of rice and harpin protein Hpa1 of Xanthomonas oryzae pv. oryzae

WANG Huan, TIAN Yi-Min, DONG Han-Song^*

College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

Corresponding author: DONG Han-Song; E-mail: hsdong@njau.edu.cn

Abstract:

In this study, we generated mutant versions of rice (Oryza sativa) aquaporin OsPIP1;3 by fragment deletion and site-directed mutagenesis, respectively. When a split-ubiquitin–based yeast two-hybrid system was employed to analyze protein combinations of Hpa1 (a harpin protein of Xanthomonas oryzae pv. oryzae) with the canonical form and mutant versions of OsPIP1;3, interaction was found between Hpa1 and the canonical form, but not in the combination of Hpa1 with the mutant protein generated by deleting a short intracellular region called connecting loop E and transmembrane domain 6 (TM6). Analyses of additional substitutive protein mutants indicated that residues 276–279 in TM6 were also important for the molecular interaction. These data suggest that loop E and TM6 are determinants of OsPIP1;3 interaction with Hpa1 while particular residues 276–279 play crucial roles in the molecular interaction.

Key words: Hpa1; OsPIP1;3; site-directed mutagenesis; membrane-based yeast two-hybrid; protein interaction